Rapid method to make OP detoxifying sponges composed of multiple immobilized enzymes of cholinesterases and OP hydrolases and oximes as reactivators

ABSTRACT

A material comprising a porous support and a plurality of enzymes for the removal, decontamination or neutralization of hazardous chemicals such as OP compounds is disclosed. The material may be used on a variety of surfaces, including natural, synthetic and biological surfaces such as skin and other delicate membranes. Also disclosed is a process of making the material, kits and various methods and reactivation devices for reactivating the enzymatic activity of the material.

TECHNICAL FIELD

[0001] This invention relates to materials, compositions, kits andmethods for neutralizing, detoxifying or decontaminating equipmentand/or personnel exposed to organophosphorous and organosulfurcompounds.

BACKGROUND OF THE INVENTION

[0002] Methods for decontamination, neutralization and removal ofchemicals, such as organophosphorous and organosulfur (OP refers toboth) compounds, herbicides and insecticides, are known in the art.However, the compositions and devices utilized in the prior art methodshave undesirable properties, such as corrosiveness, flammability,toxicity, difficulty in making and storing, and limited shelf-life.

[0003] For example, DS2, a standard decontamination agent, comprises 70%diethylenetriamine, 28% ethylene glycol monomethyl ether, and 2% NaOH byweight. Although DS2 is effective, it is corrosive upon exposure to air.DS2 and any matter resulting from its use is classified and regulated ashazardous material. After an application, the DS2 must stand for 30minutes before rinsing the treated area with water. Additionally, DS2comprises a teratogen.

[0004] Some decontamination methods employ hypochlorite formulationswhich are corrosive and toxic and injure humans and sensitive tissuessuch as eyes. Other methods comprise incinerating the contaminatedmaterial and utilizing carbon filters to absorb the residual chemicals.Yet other methods utilize polymer beads or microemulsions which absorbthe chemical and must be rinsed away. These methods are inherentlydangerous, expensive and generate hazardous waste. Furthermore, as manyof these compositions and compounds utilized degrade upon exposure towater and carbon dioxide, these compositions and compounds must be usedthe same day they are made.

[0005] Some in vivo methods employ cholinesterases in the presence ofnucleophilic oximes to detoxify OP compounds. This enzyme bioscavengerapproach is effective against a variety of OP compounds in rodents andnonhuman primates. For example, pretreatment of rhesus monkeys withfetal bovine serum acetylcholinesterase (FBS-AChE) or horse serumbutyrylcholinesterase (Eq-BChE) confers protection against up to 5 LD₅₀of soman, a highly toxic OP nerve agent. Although, the use of an enzymeas a single pretreatment drug for OP toxicity is sufficient to providecomplete protection to an individual subject, a relatively large(stoichiometric) amount of the enzyme is required to neutralize the OPcompound in vivo. Therefore, OP/enzyme stoichiometry is increased bycombining enzyme pretreatment with oxime reactivation so that thecatalytic activity of OP inhibited FBS-AChE is rapidly and continuouslyrestored, and the OP compound is detoxified.

[0006] Clearly, a need for better methods and devices for neutralizing,detoxifying, decontaminating and cleaning materials, equipment andpersonnel exposed to OP compounds exists.

[0007] Recently, OP detoxifying compounds, devices and methods thereof,which allow the safe, effective and convenient detoxification of highlytoxic compounds not possible by the prior art, have been developed.These environmentally friendly compounds, devices and methods aredisclosed hereinbelow.

SUMMARY OF THE INVENTION

[0008] The present invention provides materials, compositions, kits andmethods for neutralizing, detoxifying or decontaminating equipmentand/or personnel exposed to OP compounds.

[0009] In one embodiment, the invention relates to a material comprisinga mixture of enzymes and substrates for the removal, decontamination andneutralization of OP compounds including those directed against humans.The mixture of enzymes utilized comprises cholinesterases (ChEs) and/orOP hydrolases and reactivators, such as oximes which includesmono-disquarternary oximes. The material may comprise a flexible orrigid porous support. The porous support may be a polyurethane matrix orequivalent.

[0010] For example, the porous support may be a flexible sponge-likesubstance or like material, wherein the enzymes are secured byimmobilization. Depending on the polyurethane prepolymer or substrateutilized, porous supports of varying degrees of flexibility and porositymay be obtained. The porous support may be formed into various shapes,sizes and densities, depending on need and the shape of the mold. Forexample, the porous support may be formed into a typical householdsponge or a towelette. The preferred dimensions of the sponge are1″×2″×8″ to 2″×4″×8″. The preferred dimensions of the towelette are4″×4″×0.25″ to 4″×4″×0.03125″ to 14″×14″×0.0625″. However, duringlarge-scale synthesis, the dimensions of the initial immobilized enzymeproduct might be large. For example, approximately 4 feet by 8 feetrolls could be produced and sized as appropriate and described above.The sponge-like support would be preferable for use on surfaces,including natural, synthetic and biological surfaces such as equipment,laboratory hardware, devices, skin and other delicate membranes, wheredecontamination of a rough or irregular surface is desired or where theprior art decontamination materials are incompatible with human tissue.For example, the materials may be used to clean and decontaminate woundsas it is non-toxic and the immobilized enzymes will not leach into awound. Therefore, the sponges could be used to decontaminate civilianscontaminated by a terrorist attack at a public event.

[0011] If an object and/or area to be neutralized or decontaminatedcomprises cracks, crevices, porous or uneven surfaces, a foam-likesupport is suitable. Application of small quantities may be done with aspray-bottle or spray can with an appropriate nozzle. Further, foam maybe selected so that it can be dispensed into the opening of sensitiveequipment or an orifice of a subject, such as the ear canal. If a largearea is contaminated, an apparatus that dispenses a large quantity offoam may be utilized.

[0012] The foam-like support may dissipate after a period of time likeshaving cream or it may cure into a stable and flexible sponge-likesupport. The dissipating foam may be applied on living subjects. Thefoam, which cures, may be applied around an object and contain thecontamination within the foam. Once the foam cures, the object may behandled and moved without further exposure to the hazardous chemical.

[0013] When necessary, the material may also comprise a rigid and poroussupport. The rigid material can be ground into a powder and added tolotions, soaps and other liquids for application. Likewise, the flexiblematerial, supra, may be appropriately treated to render it suitable foruse in lotions, soaps and other liquids.

[0014] The material may also be in the form of a filter forneutralizing, detoxifying or decontaminating gases such as air.Additionally, the material may be in a form suitable for use as clothingor linings of clothing. Furthermore, the material may be used todecontaminate water by placing the material in water and then removingit from the water.

[0015] In another embodiment, the material can be color-coded accordingto the specific substance it may neutralize, detoxify or decontaminate.The color or color scheme could be selected to indicate enzymaticconcentration, activity and/or remaining shelf-life or range thereof.

[0016] As disclosed herein, one of ordinary skill in the art willappreciate the various materials and their uses as contemplated by theinventors. All of these forms may be appropriately combined with carbonfor further absorption of OP compounds. The carbon may be embedded orincorporated within the porous support of the material or the carbon maybe a layer, filter or other to be used in conjunction with the material.Additionally, a slow release form, such as a dry capsule, pellet,liposome or other, of a reactivating compound and OP reacting compoundssuch as certain oximes like HI-6 and mono-bisquaternary oximes such aspralidoxime chloride (2-PAM) may be embedded or incorporated within theporous support of the material.

[0017] A preferred embodiment of the invention comprises a materialwherein AChE and/or BChE are simultaneously immobilized with OPhydrolases on or within the porous support during synthesis of thematerial. Preferably, the enzymes are immobilized through covalentlinkages. The enzymes may be of prokaryotic or eukaryotic origin. Theenzymes may be contained within the cell or cell free. The enzymes maybe of recombinant origin. Other enzymes capable of hydrolyzing hazardouschemicals such as OP compounds may be employed, for example laccase.Additionally, other OP hydrolyzing enzymes would ensure rapid andcomplete destruction of any toxic intermediate (for example,phosphoryloximes) that might be generated during the decontaminaitonprocess. Likewise, enzymes such as triesterase may be used for thedecontamination of pesticides in a similar manner as herein described.Preferred enzymes are those that may be reactivated.

[0018] The materials of the invention may be placed in containers tocomplete decontamination the OP compounds on the materials.

[0019] In another embodiment, the invention relates to the process ofmaking a material, for the removal, decontamination or neutralization ofhazardous chemicals such as OP compounds, comprising a mixture ofenzymes immobilized on a porous support. In this embodiment, a mixtureof enzymes and a prepolymer are gently and evenly mixed together withminimal degradation of the biotype component so that the resultingimmobilized enzyme may effectively decontaminate, neutralize or detoxifyan amount of an OP compound. The device utilized folds the componentsinto one another. This is a low shear process. During synthesis of thematerial by prior art methods, for example a mixing drill, the enzymesutilized are subjected to fluid forces or shear stress. Use of a devicethat gently folds the components into one another greatly reduces thesefluid forces or shear stress, and is the preferred device for enzymes,specifically enzymes that are sensitive to the high shear forces of thedrill mixing device. Additionally use of additives such assurface-acting polymers, e.g. P-65, or low concentrations of glycerolprotects against enzyme denaturation induced by shear forces, andmodifies the final properties of the sponge to obtain the desiredporosity and absorbent qualities.

[0020] In a preferred process of making the material, a two chamberapparatus is utilized. See FIGS. 11A and B. One chamber contains amixture of enzymes and the other chamber contains the prepolymer. Themixture of enzymes and the prepolymer are simultaneously extruded at a1:1 ratio and mixed. Preferably, the mixture of enzymes and theprepolymer are rapidly and evenly extruded through a static mixingstator which gently and evenly mixes the enzymes and prepolymer. Apreferred low shear device is a double chamber syringe and a staticmixing stator typically used to mix viscous polyurethanes or epoxyglues. The size of the apparatus may vary depending on need. It may bepocketsize for use in the field by soldiers. Alternatively, theapparatus may be suitable for large-scale production and/ordecontamination of a large objects or area. The low shear mixing devicemore than doubles the resultant AChE or BChE immobilized enzyme activitywhen compared to an identical mixture prepared with the high sheardevice.

[0021] The invention further relates to various materials, methods anddevices for reactivating the enzymatic activity of the material. Thesematerials, methods and devices will allow a person to use thedecontamination material of the invention for several separate usesand/or for a single and continuous use, which would normally requireseveral decontamination materials but for reactivation of the enzymaticactivity of the immobilized enzymes. Additionally, these materials,methods and devices allow for complete decontamination and/orneutralization of excess OP compounds absorbed by the porous support butdid not react with the immobilized enzymes. These methods andreactivation materials employ substrates and/or oximes, to reactivatethe catalytic activity of the OP inhibited and immobilized enzymes.

[0022] The invention further relates to various materials and additivesthat are added to the embodiment to aid in the removal anddecontamination of organophosphate from surfaces such as cracks,crevices, porous or uneven surfaces such as clothes and biologicalsurfaces that readily absorb the organophosphates or pesticides such asskin. The additives are used in conjunction with the sponge material andmay be incorporated within the porous support of the material. Theadditives may be in a dry or liquid form, and may be organophosphatesolubilizing compounds such as triacetin or tetraglyme, or oximes, whichboth aid in decontaminating and reactivating enzymes.

[0023] Another embodiment of the invention relates to a variety of kits.Along with the sponge containing a plurality of enzymes needed for thedecontamination of organophosphorous and/or sulfur compounds the kit mayinclude materials which would facilitate or be deemed necessary for thedecontamination process. Kits may also include polymeric materials andenzymes if the foam is transient in nature, e.g. the prepolymer, astable enzyme mixture and a low shear apparatus for making anorganophosphorous and/or organosulfur decontamination foam. The kits maycontain items to facilitate the use of the device, e.g. instructions,containers, test tubes, etc.

DESCRIPTION OF THE DRAWINGS

[0024] This invention is further understood by reference to the drawingswherein:

[0025]FIG. 1A illustrates the modeled surfaces of acetylcholinesterase,butyrycholinesterase and phosphotriesterase.

[0026]FIG. 1B illustrates the modeled surface of laccase.

[0027]FIG. 2 shows a cured material.

[0028]FIG. 3 schematically illustrates the specific reaction of theenzymes with prepolymer and the product polyurethane crosslinked ChE.

[0029]FIG. 4 shows the linear correlation between the amount of BChEadded during synthesis of the material and the amount of BChE in thefinal material.

[0030]FIG. 5 shows the increasing amounts of BSA added during synthesisto a constant amount of AChE and TDI polymer.

[0031]FIGS. 6A and B illustrates that the materials maintained enzymaticstability for more than 12 months at 25° and 45° C. and 3 years at 4° C.for AChE(A) and BChE(B).

[0032]FIG. 7 shows that the material maintained enzymatic activity afterconsecutive washes.

[0033]FIG. 8 shows a substrate concentration dependent curve for solubleand polyurethane coupled AChE.

[0034]FIG. 9 illustrates the pH range of soluble and immobilized AChE.

[0035]FIG. 10 shows the relative activities of co-immobilized ChEs andOPHs.

[0036]FIGS. 11A and B shows a version of a manual mixing gun and adisposable mixing stator.

[0037]FIG. 12 illustrates how oximes reactivate alkylphosphorylated ChE.

[0038]FIG. 13A illustrates the enzyme activity of immobilized FBS-AChE.FIG. 13B illustrates the enzyme activity of immobilized Eq-BChE.

[0039]FIG. 14 represents inhibition of foam-immobilized FBS-AChE by DFPand reactivation by HI-6.

[0040]FIG. 15 represents inhibition of foam-immobilized Eq-BChE by DFPand reactivation by TMB4.

[0041]FIG. 16 shows restoration of the original sponge activity withHI-6.

[0042] FIGS. 17 (A, B and C) shows the protection afforded by spongewith various additives, i.e. tetraglyme (A), HI-6 (B) and 2-PAM (C).

[0043]FIG. 18 shows the capacity of the resulting carbon sponge forbinding methylene blue.

DETAILED DESCRIPTION OF THE INVENTION

[0044] Enzymes have been incorporated in hypo-based urethane foam duringpolymer synthesis. See U.S. Pat. No. 4,342,834. Hypoprepolymer issynthesized from a reaction of polyether (or polyester) polyol withisocyanates in the presence of cross-linking agents. See Havens, P. L.,et al., Ind Eng Chem Res (1993) 32:2254-2258; U.S. Pat. No. 4,137,200;LeJeune, K. E., et al., Biotechnology and Bioengineering (1999)20;62(6):659-665. Synthesis is initiated by bringing water moleculesinto contact with isocyanate groups present within the polyurethaneprepolymer.

[0045] A two-step procedure occurs from this point. Isocyanates reactwith water to form an unstable carbonic acid, which in turn degrades toan amine yielding CO₂ that gives the porous support lift and enables itto rise. The amines readily react with isocyanate groups, leading toproduction of urea type linkages. Since the enzyme contains multiplefunctional groups, such as amines and hydroxyls that can react withisocyanates, the enzyme becomes an integral part of the porous supportduring synthesis. Significant quantities of enzyme can link to theporous support without disrupting the progress of polymer synthesis. Thereaction occurring during the polymer synthesis is shown below.

[0046] 1. CO₂ Evolution:

[0047] 2. Urea Linkage:

[0048] 3. Amine Group Enzyme Immobilization:

[0049] 4. Hydroxyl Group Enzyme Immobilization:

[0050] The following list of enzymes and chemicals are examples of thosesuitable for use in the instant invention:

[0051] Acetylcholinesterase (AChE);

[0052] Butyrylcholinesterase (BChE);

[0053] Pseudocholinesterase;

[0054] Organophosphate hydrolases (OPH);

[0055] Phosphotriesterase;

[0056]Pseudomonas diminuta bacterial OPH (paraoxonase);

[0057] Laccases;

[0058] Organophosphate acid anhydrase (OPAA)

[0059] Pralidoxime chloride (2-PAM);

[0060] 7-(methoxyphosphinyloxy)-1-methylquinolium iodide (MEPQ);

[0061] Diisopropyl fluorophosphate (DFP);

[0062] Acetylthiocholine iodide (ATC);

[0063] S-butyrylthiocholine iodide (BTC);

[0064] 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB);

[0065] N,N′-trimethylene bis(pyridinium-4-aldoxime)dibromide (TMB4); and

[0066]1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxyaminopyridinium)-dimethyletherhydrochloride (HI-6).

[0067] The following examples are intended to illustrate but not tolimit the invention.

EXAMPLE 1

[0068] Determination of Possible Enzyme Interference

[0069] As polyether prepolymer derived from tolyl diisocyanate (TDI),reacts most favorably with free aliphatic amines such as lysine andarginine present on the surface of the ChEs (or any protein) to become apermanent cross-linked part of the material, computer aided molecularmodeling of the enzymes was performed to highlight the available aminogroups on the surface of each enzyme, and to determine whether thecoupling of these groups to a porous support would interfere withenzymatic function. This may be performed on every enzyme for which itscrystal structure is known, or enzymes which may be modeled by homology.

[0070]FIG. 1A illustrates the modeled surfaces of acetylcholinesterase,butyrycholinesterase and phosphotriesterase and shows the lysine andarginine residues on the surface of the ChEs which are available forcoupling to the prepolymer. This was generated by Insight II, molecularmodeling software, by Biosym Technologies. Based on the molecularmodeling, there are at least one lysine and 29 arginine water-accessibleresidues on the surface of FBS-AChE to couple to the porous support,while 26 lysine and 26 arginine residues were modeled for equine-BChE.The majority of the lysine and arginine residues were found on thebackside of the ChEs, and only a few are found on the side of the enzymewhere the catalytic site gorge is located. The rim and the catalyticsite gorge opening of both AChE and BChE appeared to be essentiallydevoid of lysine and arginine. Therefore, coupling these enzymes to theporous support should have minimal effect on the entrance of substrate,inhibitors such as OPs, or reactivators such as oximes which includesmono-disquarternary oximes, release of products of catalysis to and fromthe active site, and the kinetic rates of the enzymes. Similarly, amodel of the surface of laccase (FIG. 1B) is shown with availableresidues to couple covalently to the prepolymer.

EXAMPLE 2

[0071] Synthesis of an Enzyme Bound Polyurethane Material

[0072] A typical synthesis of the material comprises mixing enzymes inphosphate buffer containing 1% (final concentration) surfactant withprepolymer. Polyether prepolymer derived from tolyl diisocyanate (TDI),Hypol prepolymer TDI 3000 (Hampshire Chemical, Lexington, Mass.), andPluronic P-65 surfactant (BASF Specialty Chemicals, Parsippany, N.J.)were used. The 2-phase system is mixed and placed into a suitable moldand left to cure. FIG. 2 shows a cured material which comprises asponge-like support.

[0073]FIG. 3 schematically illustrates the specific reaction of theenzymes with prepolymer. Synthesis begins when H₂O molecules react withthe isocyanate groups present within the polyurethane prepolymer.Isocyanate reacts with the water to form an unstable carbonic acid,which degrades to an amine yielding CO₂. The CO₂ causes the polymer torise and become porous, and simultaneously the amines readily react withthe isocyanate groups leading to urea linkages. While the amino groupsare the preferred reaction site between the enzyme(s) and theprepolymer, hydroxyl (OH) groups are also available to interact withinthe reaction buffer, e.g. H₂O and on the enzyme(s).

[0074] Since the ChE contains amines that are on the surface andavailable to react with the isocyanate groups, they can become anintegral part of the polyurethane support during synthesis. There is nosignificant entrapment of the enzyme in the material as found withcyclodextrins, or physical adsorption of the enzymes, as observed withactivated carbon. The inclusion of a surfactant such as Pluronic P-65 atabout 1% final concentration controls the final structure and absorptionpotential of the material.

[0075] To create a material comprising a porous polyurethane support,approximately 30 mL of 50 mM phosphate buffer, pH 8.0, containing P-65surfactant buffer, was placed in a 600 mL plastic beaker. 3 to 5 mL ofeither purified FBS-AChE (7500 units) or purified Eq-BChE (5000 units)was added, followed by approximately 40 gm of Hypo 3000 prepolymer(tolyl diisocyanate). The two-phase system was mixed and the materialwas allowed to expand for 10 min, extruded from the container. Thematerial was washed thoroughly with 50 mM phosphate buffer, pH 8.0,dried and stored in a zippered bag at 4° C. for future use.

EXAMPLE 3

[0076] Characteristics of Synthesized Material

[0077] Approximately 20-90% of the enzymes were covalently linked to theporous support through free amino- or hydroxyl groups. This wasdetermined by the presence of enzyme in first and second washes of thematerial.

[0078] Since the enzymes can be attached at multiple points, they becomea part of the cross-linked polymer support. The cross-linked polymersupport imparts considerable stability to the bound enzymes. A largequantity of enzyme can be incorporated into a small polyurethanesupport, thereby rendering the cross-linked polymer support a highlyeffective material for decontamination.

[0079] A. Enzymatic Activity.

[0080] Five samples of materials containing FBS-AChE and five samples ofmaterials containing Eq-BChE, ranging in weight from 1 to 40 mg, weresuspended in 2.8 mL of 50 mM phosphate buffer, pH 8.0, and assayed usingthe method of Ellman. See Ellman, G. L., et al., (1961) BiochemPharmacol. 7:88-95. A linear correlation was found between the weight ofthe sponge and enzyme activity for both FBS-AChE and Eq-BChEimmobilizations. See FIGS. 13A and B. The linear correlation between theweight of the material and enzyme activity indicates a uniformimmobilization of AChE or BCHE throughout the material.

[0081] The material was washed with either 50 mM phosphate buffer,distilled water, or 10 mM ammonium bicarbonate without affectingsubstrate hydrolysis. Therefore, the mixing of prepolymer, surfactant,and enzyme in situ at 22° C. yields a useful and effective materialretaining about 50% of the original activity of soluble ChE.

[0082] B. Protein Loading Capacity.

[0083] The material has a significantly higher loading capacity for ChEssuch as BCHE or AChE. The final activity of the BCHE immobilized in thematerial could be increased by adding larger quantities of enzyme duringsynthesis. See FIG. 4. When nonspecific protein (bovine serum albumin,BSA) was added to a constant amount of purified AChE, there was noreduction in ChE activity. See FIG. 5. Thus, higher potency materialsmay be synthesized with additional proteins, enzymes and other ChEs.Additionally, materials effective against a diverse array of OPcompounds may be readily synthesized by with combinations of multipleenzymes or a plurality of enzymes.

[0084] C. Enzymatic Stability.

[0085] As illustrated by FIG. 6, the immobilized ChE and OP hydrolasemaintained enzymatic stability for more than 12 months at 25° C. and 45°C., respectively. If the material is frozen in liquid nitrogen, most ofthe original activity remains. TDI imparts remarkable stability to theimmobilized ChE; about 50% of the original activity of the immobilizedAChE and 20% of the activity of the immobilized BChE remained after 16hours at 80° C., conditions under which the soluble enzymes wouldexhibit no activity. The ChE materials can be exhaustively dried undervacuum at 22° C. and then rehydrated without loss of enzyme activity.When AChE or BChE materials were exhaustively washed and assayed foractivity, the wash and assay cycle repeated more than twenty times overthree days, no decrease in activity occurred. See FIG. 7. This indicatesthat the material may be used repeatedly.

[0086] These results also demonstrate that the ChEs are covalentlycross-linked in the porous support and that the ChEs will not leach outto skin, water, or equipment. Therefore, once the immobilized enzymesbind an OP compound the OP is removed from the surface requiringdecontamination.

[0087] D. Kinetic Constants.

[0088] The number of active sites of either the immobilized or solubleChEs was determined by titration with the organophosphorous compoundMEPQ, 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide. Thebimolecular rate constants for the inhibition of AChE material and BChEmaterial and the respective soluble enzymes by MEPQ at 25° C. showedthat there was no significant difference between the soluble andcovalently bound enzymes. See Table 1. These TABLE 1 Time-DependentInhibition of ChEs by MEPQ Enzyme Bimolecular rate constant ChE Form(M⁻¹ min⁻¹) ± SD FBS-AChE soluble 1.59 ± 0.52 × 10⁸ coupled to sponge1.00 ± 0.28 × 10⁸ Equine-BChE soluble 4.15 ± 0.78 × 10⁷ coupled tosponge 4.21 ± 2.00 × 10⁷

[0089] results demonstrate that the immobilized and soluble forms ofChEs interact with the OP compounds similarly. Therefore, enzymaticactivity assays which are generally available and known in the art maybe used.

[0090] An initial rates method using a modified Ellman's assay was usedto determine the parameters K_(m), k_(cat), and k_(cat)/K_(m) forimmobilized and soluble AChE and BChE. The number of active sites ofeither the coupled or soluble ChEs was determined by titration withMEPQ. As shown in Table 2 and FIG. 8 for AChE, the K_(m) values for theimmobilized ChEs were about 10-fold greater than the correspondingsoluble enzymes, and the k_(cat) values were less dramatically affected.The combined effects on affinity for substrate and k_(cat) resulted inapproximately a 20 to 50-fold decrease in acylation (k_(cat)/K_(m)).Interestingly, while soluble BChE lacked substrate inhibition,immobilized BChE yielded substrate inhibition. These results suggestthat covalent binding of surface residues of ChEs to the porous supportchanged some properties of the active site region of the bound enzymesdirectly or indirectly. TABLE 2 Kinetic parameters for soluble andpolyurethane coupled ChEs. Substrate K_(m) K_(ss) K_(cat) K_(cat)/K_(m)Enzyme Form inhibition (mM) (mM) B (min⁻¹) (M⁻¹min⁻¹) FBS-AChE Solubleyes 0.119 18 — 2.8 × 10⁵ 2.5 × 10⁹ immobilized yes 1.090 22 — 5.9 × 10⁴5.4 × 10⁷ Equine-BChE Soluble no 0.127 1.5 1.8 3.1 × 10⁴ 2.4 × 10⁸immobilized yes 1.200 16 — 1.8 × 10⁴ 1.5 × 10⁷

[0091] Generally, immobilized cholinesterases or OP hydrolyzing enzymesexhibit between the same to 10 fold greater K_(m) values than thecorresponding soluble enzymes. In addition to the cholinesterases, OPH(derived from Pseudomonas diminuta, FIG. 19A) shows about a 10-foldincrease in K_(m) because a shift to the right is also observed in theimmobilized (sponge) form when determined using the substrate paraoxon.On the other hand, OPAA (derived from Alteromonas, FIG. 19B), showslittle change in K_(m) for the substrate paraoxon.

[0092] E. pH of Soluble and Immobilized Enzymes.

[0093] The pH profiles of immobilized and soluble AChE are identical andthe enzymes exhibit activity throughout the broad pH range of 7-8.5. SeeFIG. 9. Since the pH profiles of soluble cholinesterases and OPhydrolases have optimal activities in this same pH range, the materialsmay be optimized and diversified by employing a plurality of thesemultiple enzymes immobilized on or within a porous support.

EXAMPLE 4

[0094] Immobilization of a Plurality of Enzymes

[0095] ChEs were co-immobilized with bacterial OP hydrolase (OPH_(B))and/or rabbit serum OP hydrolase (OPH_(R)). There was no reduction inthe enzymatic activities of AChE or BChE co-immobilized with OPH ascompared to the enzymatic activities of each of these enzymesindividually immobilized. See FIG. 10. Additionally, there was noreduction in the enzymatic activity of co-immobilized OPH. Therefore, aplurality of enzymes, which each enzyme differentially reacts withvarious OP compounds, may be selected and utilized in a material tocreate a decontamination material effective against a wide range of OPcompounds.

EXAMPLE 5 Rapid Mixing Synthesis

[0096] By utilizing a method of syntheses modified from the adhesiveindustry (CPA, Greenville, R.I. 02828) shear forces which decreaseenzymatic activity are reduced. See FIG. 11. In this method, the enzymeis not in an organic buffer as required in some immobilizationtechniques. This results in less air-induced shearing, therebymaintaining enzymatic activity. This method is also simple to conduct,rapid and reproducible. The low shear mixing device more than doublesthe resultant AChE and/or BChE immobilized enzyme activity when comparedto an identical mixture prepared with the high shear device such as amixing drill. See Table 3. TABLE 3 Technique AChE Activity U/mg Highshear mixing drill 0.100 Low shear 2-chamber device 0.270

EXAMPLE 6

[0097] Inhibition of Immobilized FBS-BChE with DFP and Reactivation withHI-6

[0098] 100 mg samples of immobilized FBS-AChE were incubated withvarying concentrations of DFP in 2 mL of 50 mM phosphate buffer, pH 8.0,for 1 hour at 25° C. In parallel experiments, 1 mM HI-6 was added to thesame amount of material and DFP. Residual DFP in the samples wasmeasured by adding a 0.5 mL aliquot of the reaction mixture to 0.5 mL ofa fresh 1 U/mL solution of FBS-AChE, incubating for 1 hour, and assaying10 T¹ aliquots using the Ellman procedure. The results are shown in FIG.14.

[0099] The inhibition of FBS-AChE activity by DFP was proportional tothe stoichiometric amount of DFP added to the foam suspended in buffer.The presence of 1 mM HI-6 nearly completely prevented enzyme inhibitionby DFP. This indicates that immobilized FBS-AChE may be repeatedlyreused after reactivating the enzyme with an oxime solution such asHI-6.

[0100]FIG. 12 illustrates how oximes may reactivate alkylphosphorylatedChE activity.

EXAMPLE 7

[0101] Inhibition of Immobilized Eq-BChE with DIP and Reactivation withTMB4

[0102] 50 mg samples of immobilized Eq-BChE were incubated with varyingconcentrations of DFP in 2 mL of 50 mM phosphate buffer, pH 8.0, for 18hours at 25° C. In parallel experiments, 1 mM TMB4 was added to the sameamount of material and DFP. Residual DFP in the samples was determinedby adding a 0.5 mL aliquot of the reaction mixture to 0.5 mL of a fresh1 U/mL solution of Eq-BChE, incubating for 1 hour, and assaying 10 T¹aliquots using the Ellman procedure.

[0103] TMB4 was used as a reactivator instead of HI-6, since TMB4 is amore efficient reactivator of inhibited Eq-BChE than is HI-6. Theseresults are shown in FIG. 15. As in Example 6, the foam-bound Eq-BChEmay be repeatedly reused after reactivating the enzyme with an oximesolution such as TMB4.

EXAMPLE 8

[0104] Inhibition of Immobilized AChE with the Organophosphate MEPQ andDetoxification of the MEPQ and Reactivation of the Immobilized Enzyme inthe presence of HI-6

[0105] 50 mg samples of immobilized acetylcholinesterase were incubatedwith varying concentrations of MEPQ in 2 mL of 50 mM phosphate buffer,pH 8.0 at 25° C. for 1 hr. In the absence of oxime HI-6, the spongesoaks up the MEPQ and is inactivated. Addition of HI-6 reactivates thesponge's activity, and the MEPQ is detoxified, and most of the originalactivity of the sponge returns. Only at very high ratios oforganophosphate (1000-fold molar excess) is the process of binding,reactivation, and detoxification not complete. However, fresh HI-6 canrestore most of the original activity once again. See FIG. 16.

EXAMPLE 9

[0106] Additives to the Sponge to Improve Decontamination of Soman (GD)Contaminated Skin of Guinea Pigs

[0107] Sponges approximately 1½×2½×¼″ (H×L×D) contained 9.0 mL ofadditive and a second sponge contained 4.5 mL of additive. Each guineapig was wiped with the first sponge and then the second sponge aftersoman (GD) exposure. Survival of the guinea pigs was determined after 24hours, and the protective ratio determined. The protective ratio is theratio of the LD₅₀ of the sponge containing an additive to the LD₅₀ ofsoman in the absence of sponge. Thus, the higher the LD₅₀, then thehigher the protective ratio and the more effective the spongecombination is for decontamination of guinea pig skin and protecting theanimal from the organophosphate. The sponge was compared to the M291 kit(available from Truetech, Inc.), the currently used decontamination kitfielded by the US Army. As shown in the table, the sponges provide 4 to5-fold better protection than the M291 kit.

[0108]FIG. 17A shows the protection afforded by tetraglyme; FIG. 17B theprotection afforded by HI-6, and FIG. 17C the protection afforded by2-PAM. The number on the top of each bar shows the number of guinea pigsevaluated at the indicated dose of soman (GD). For reference, the LD₅₀of soman on guinea pigs without any effort to decontaminate is shown bythe label “GP”, while the protection offered by the M291 kit is shown by“M291”. Other additives to the sponge such as triacetin also affordedsome additional protection compared to the M291 kit. Additive to spongeLD₅₀ Protective Ratio HI-6 (oxime, 50 mM) 79 8.0 2-PAM (oxime, 50 mM) 767.7 Tetraglyme (30%) 88 8.9 Reference values M291 decon kit 17.7 1.8Soman alone 9.9 —

EXAMPLE 10

[0109] Activated Carbon Containing Sponge

[0110] 0.5-1 grams of activated carbon was added to about 4 mL of theprepolymer prior to mixing with acetylcholinesterase (5 mL of Electriceel, in 50 mM pH 8.0 phosphate buffer with 1% Pluronic P-65) to producean acetylcholinesterase immobilized carbon sponge. The addition ofcarbon did not interfere with the immobilization of the enzyme, as shownin the table. The capacity of the resulting carbon sponge for bindingmethylene blue (an calorimetric indicator for activated carbon or resin)is illustrated in the FIG. 18. Therefore, comparison of the sponge withactivated carbon to the sponge lacking activated carbon demonstratesthat it can bind about 2-fold more methylene blue at less thansaturating concentrations. Activities of Sponges and Activated CarbonRelative Activity (% control Relative Activity to absorb Type of Spongein absence of carbon) methylene blue Electric eel AChE sponge 100 %  1 XElectric eel AChE sponge 108 %  2 X with Activated Carbon ActivatedCarbon not in the — 13 X sponge

[0111] Incorporation by Reference

[0112] To the extent necessary to understand or complete the disclosureof the present invention, all publications, patents, and patentapplications mentioned herein are expressly incorporated by referencetherein to the same extent as though each were individually soincorporated.

1. A polymeric sponge or foam for the removal, decontamination,detoxification and/or neutralization of a hazardous compound comprisinga plurality of enzymes immobilized on a polymeric porous support, saidplurality capable of detoxifying organophosphorous and/or organosulfurcompounds.
 2. The polymeric sponge or foam of claim 1 wherein saidplurality of enzymes comprises at least one enzyme selected from thegroup consisting of: acetylcholinesterase (AChE), butyrylcholinesterase(BChE), triesterase, pseudocholinesterase, organophosphate hydrolase(OPH), phosphotriesterase, paraoxonase and OP hydrolyzing enzymes. 3.The polymeric sponge or foam of claim 1 wherein said porous supportcomprises polyurethane.
 4. The polymeric sponge or foam of claim 1further comprising carbon embedded or integrated on or in the poroussupport.
 5. The polymeric sponge or foam of claim 1 further comprising areactivation compound, material or device embedded or integrated on orin or attached to the porous support.
 6. The polymeric sponge or foam ofclaim 5 wherein said reactivation compound, material or device comprisesHI-6, TMB4, or mono-bisquarternary oximes.
 7. A method of making apolymeric sponge or foam for the removal, decontamination,detoxification and/or neutralization of a hazardous compound comprisingimmobilizing a plurality of enzymes immobilized on a polymeric poroussupport, said plurality capable of detoxifying organophosphorous and/ororganosulfur compounds.
 8. The method of claim 7 wherein the pluralityof enzymes comprises at least one enzyme selected from the groupconsisting of: acetylcholinesterase (AChE), butyrylcholinesterase(BChE), triesterase, pseudocholinesterase, organophosphate hydrolase(OPH), phosphotriesterase, paraoxonase and OP hydrolyzing enzymes. 9.The method of claim 8 wherein the step of immobilizing comprises mixingsaid mixture with a polyurethane prepolymer.
 10. The method of claim 9wherein said polyurethane prepolymer comprises a diisocyanate.
 11. Themethod of claim 10 wherein the diisocyanate is tolyl diisocyanate. 12.The method of claim 9 wherein equal parts of said plurality and saidpolyurethane prepolymer are simultaneously mixed.
 13. The method ofclaim 9 wherein the step of mixing is conducted with an apparatus havinga first chamber and a second chamber.
 14. The method of claim 13 whereinsaid first chamber contains said plurality and said second chambercontains a polyurethane prepolymer.
 15. The method of claim 14 whereinsaid mixing comprises extruding equal parts of said plurality and saidpolyurethane prepolymer rapidly and evenly through a static mixingstator.
 16. A polymeric sponge or foam for the removal, decontamination,detoxification and/or neutralization of a hazardous compound made by themethod of any one of claims 7-15.
 17. A method of reactivating apolymeric sponge or foam of claim 1 comprising contacting said polymericsponge or foam with at least one compound selected from the groupconsisting of: HI-6, TMB4, and mono-bisquarternary oximes.
 18. A methodfor treating a contaminated surface comprising, contacting the surfacewith the sponge of claim 1 to detoxifying organophosphorous and/ororganosulfur compounds present on the surface.
 19. The method of claim18 further comprising contacting the sponge with an oxime.
 20. Themethod of claim 19, wherein the oxime is HI-6 or 2-PAM.
 21. The methodof claim 18, wherein the sponge additionally contains activated carbonand/or resin.
 22. The method of claim 21, wherein the sponge containsactivated carbon.
 23. A kit for the removal, decontamination,detoxification and/or neutralization of a hazardous chemical comprisinga polymeric sponge or foam of claim
 1. 24. A kit according to claim 23further comprises a compound for the reactivation of enzyme in an amountsufficient to displace covalently attached organophosphorous compoundsfrom the enzyme active site by nucleophilic attack.